Chitosan is a non-toxic cationic copolymer of N-acetyl-D-glucosamine and D-glucosamine that possesses favorable mucosal adhesion properties and has been widely used in controlled drug delivery. The mucoadhesivity of chitosan is thought to prolong the residence time of an associated drug in the gastrointestinal tract, thereby increasing its bioavailability. (Kotze A F, Luessen H L, Thanou M, Verhoef J C, de Boer A G, Juninger H E, Lehr C M. Chitosan and chitosan derivatives as absorption enhancers for peptide drugs across mucosal epithelia. In: Mathiowitz E, Chickering D E, Lehr C M, eds. Bioadhesive Drug Delivery Systems. New York, N.Y.: Marcel Dekker; 1999.)
Several groups have explored the potential of chitosan as a DNA delivery vehicle, and the properties of a number of chitosan/DNA complexes have been examined in an attempt to identify compositions well suited for gene transfection. The complexes have been found to vary in, among other properties, solubility, propensity for aggregation, complex stability, particle size, ability to release DNA, and transfection efficiency. Chitosans of large molecular weight are relatively insoluble at physiological pH and are dissolved in acidic solution for use. Once formed, complexes containing large molecular weight chitosan and DNA are relatively stable, but poor transfection efficiencies have been reported, possibly owing to poor uptake and release of DNA. Low molecular weight chitosan/DNA complexes are more soluble but less stable in solution. Chitosan polymers of low molecular weight reportedly form unstable complexes with DNA, as indicated by separation in an electric field (agarose gel electrophoresis). Such complexes also show low transfection efficiencies and low levels of reporter gene expression in vitro (e.g., Koping-Hoggard et al., Gene Ther., 8:1108-1121, 2001; MacLaughlin, et al., J Control Release. 1998 Dec. 4; 56(1-3):259-72; Sato et al., Biomaterials, 22:2075-2080, 2001; US 2005/0170355; US 2005/0164964). Experiments have also shown that low molecular weight chitosan/DNA complexes tend to release DNA in response to challenge with salt or serum, suggesting they are poorly suited to many in vivo applications.
Studies of the effect of chitosan molecular weight on transfection efficiency in vitro have been equivocal. Some studies have shown no significant dependence on molecular weight for chitosan polymers in the size range of 20-200 kDa (Koping-Hoggard et al., supra; MacLaughlin et al, supra). However, others (Sato et al., supra) have reported that chitosans of 15 kDa and 52 kDa show higher reporter gene expression in vitro than chitosan polymers>100 kDa and chitosan polymers of 1.3 kDa are ineffective. Moreover, discordance between in vitro and in vivo transfection efficiencies has been frequently reported (e.g., Koping-Hoggard et al., Gene Ther. 2004 October; 11(19):1441-52; US 2005/0170355; US 2005/0164964).
A number of studies have examined the ability of large molecular weight chitosan/DNA complexes to transduce cells of the gastrointestinal tract. Chitosan/DNA complexes incorporating an endosomolytic peptide have been administered directly to the upper small intestine and colon have been shown to produce reporter gene expression in epithelial cells, Peyer's patches and mesenteric lymph nodes (MacLaughlin et al., supra). Additionally, oral administration of a chitosan complex with DNA encoding erythropoietin has been shown to produce a transient hematocrit increase (Chen et al., World J. Gastroenter., 10:112-116, 2004). Chitosan has also been used in a food allergy model to orally deliver DNA encoding a peanut allergen protein, Arah2, in an attempt to render mice tolerant to the ingestion of peanut extract (Roy et al., Nat. Med., 5:387-391, 1999). In all, relatively low transfection efficiencies and low levels of transgene expression have been reported, due in part to poor DNA uptake and release, but also due in part to rapid turnover of cells in the gut. The gut epithelium is one of the most rapidly renewing tissues in the body, with epithelial cell turnover every 3-5 days. Importantly, transgene expression in mucosal cells of the gut is additionally complicated by the fact that luminal mucosal cells are short-lived, providing a brief period of time for expression of DNA once it has entered the nucleus of a cell.
Other research groups have chemically modified chitosan in a variety of ways in efforts to develop DNA complexes with improved transfection efficiencies and other desirable properties, such as the ability to transduce distal gut tissue. Kai et al. point out in their report that chitosan/DNA compositions that have left the stomach and entered the more neutral environment of the duodenum lose positive charge with the attendant shift in pH, and consequently tend to release associated DNA. Kai et al. report that N-acetylation of lyophilized large molecular weight chitosan/DNA complexes stabilizes orally delivered complexes and increases efficiency of distal gut transduction. Kai et al., Pharm. Res. 21:838-843, 2004.
Given the lifespan of luminal gut mucosal cells, it is perhaps not surprising that long-term expression of genes delivered to gut mucosa by chitosan has not been reported. Additionally, the ability of chitosan DNA complexes to transfect less prevalent cell types of the gut mucosa, such as endocrine cells, has not been examined in detail. The difficulty associated with achieving long-term expression and transfection of gut endocrine cells may be appreciated by a consideration of gut structure.
The wall of the gastrointestinal canal is composed of four layers. The innermost layer of the canal is the mucosal layer, which is composed of a lining epithelium that borders the lumen. The epithelium is the site at which the body interacts with ingested materials. In areas of the gastrointestinal canal where absorption is effected, the epithelium is a single cell in thickness. The epithelium rests on a basal lamina, which in turn overlies the lamina propria. Beds of blood capillaries are densely packed in the length of lamina propria underlying absorptive regions of the canal, and it is into these vessels that the processed products of absorbed food matter pass.
The human small intestine consists of three portions: duodenum, jejunum, and ileum. The mucosa of the small intestine is extensively folded giving it a ruffled appearance as circular folds project into the intestinal lumen. Such folding fills a substantial area of the intestinal canal and increases the absorptive surface area of the epithelium by several fold. At the luminal surface, the folds of the intestinal mucosa present villi, which are evaginations of the mucosa that further increase absorptive area. Each villus, in turn, is covered by an epithelium one cell thick. This epithelium is overwhelmingly populated by absorptive enterocytes, which display thousands of short microvilli on their apical (luminal) surface, increasing the absorptive area many fold again. The outer surface of the microvilli, referred to as the glycocalyx, is filamentous and rich in carbohydrates. This membrane region is also rich in a wide variety of enzymes and transport systems facilitating the breakdown and uptake of ingested material.
Absorptive enterocytes constitute greater than 90% of the epithelial cells of the villus, and an even greater proportion of the luminal surface area. Scattered among these cells are the relatively small number of enteroendocrine cells, which reportedly constitute approximately 0.3% of the villus epithelium. In contrast to absorptive enterocytes, which present a large and ultrastructurally complex apical surface, endocrine cells have broad basal surfaces juxtaposed to capillaries of the lamina propria and narrow superiorly toward the lumen.
Even more elusive than the endocrine cells of the gut mucosa are the gut mucosal precursor cells. Inferior to the projecting villi, in the epithelium lining the depth of the crypts, lie precursor cells that give rise to the major cell types of the mucosa, including absorptive enterocytes and gut endocrine cells. The villus epithelial layer forms a continuous sheet of short-lived differentiated epithelial cells that is renewed about every three days, and maintenance of the epithelium requires an enormous amount of cell division and differentiation. Precursor cells of the crypts generate progeny that migrate out of the crypts toward villi and undergo differentiation.
Gut endocrine cells are generally characterized by their ability to secrete a synthesized protein into the blood in response to a signal or stimuli (a “secretagogue”). Particular examples of endocrine cells include K cells, L-cells, S-cells, G-cells, D-cells, I-cells, Mo-cells, Gr-cells.
K cells are located primarily in the stomach, duodenum, and jejunum. These endocrine cells secrete the hormone GIP, which normally functions to potentiate insulin release after a meal.
Gut endocrine cells generally, and K cells in particular, are attractive cellular targets for the delivery of transgenes. These cells possess the ability to process proforms of many proteins, and possess the cellular machinery that provides for regulated secretion of protein into the systemic circulation in response to cues. These properties have been exploited previously (see Cheung et al., Science, 290:1959-1962, 2000; U.S. patent application Ser. No. 09/804,409; expressly incorporated herein by reference). K cells engineered with a K-cell specific glucose-responsive insulin expression construct were observed to express and secrete insulin in response to elevated blood glucose, and were capable of restoring normal glucose tolerance in a mouse model of diabetes.
Despite a general interest in chitosan as an alternative to viral means of nucleic acid delivery, low transfection efficiency, stability and solubility problems, in vivo unpredictability, and an inability to transfect other than short-lived mucosal cells has largely prevented application of chitosan as a nucleic acid carrier in the harsh environment of the gut.